A Two-Step Synthesis of Unprotected 3-Aminoindoles via Post Functionalization with Nitrostyrene

A novel, low-cost method for the preparation of not easily accessible free 3-aminoindoles has been developed. This approach is based on a well-established reaction between indoles and nitrostyrene in the presence of phosphorous acid, which results in the formation of 4′-phenyl-4′H-spiro[indole-3,5′-isoxazoles]. The latter could be transformed to corresponding aminated indoles by reaction with hydrazine hydrate in good or excellent yields upon microwave-assisted heating.


Introduction
3-aminoindolic motif commonly occurs in many natural and artificial substances demonstrating a wide range of biological activities [1][2][3][4][5]. Ideally, to install this fragment into the target molecules, one would need a convenient, broadly applicable synthetic pathway to free, 3-aminated indoles as corresponding precursors or building blocks. However, the instability of the unprotected, electron-rich 3-aminoindoles, which are sensitive to light and air and tend to undergo oxidative dimerization and/or other types of decomposition reactions [6][7][8] is the main reason why such a method is still yet to be found. Until then, most of the reported synthesizes toward 3-aminoindoles are usually dealt with relatively stable electron-pour-deactivated derivatives [9][10][11][12] or rely on capping the in situ generated amino group with suitable protective groups [13][14][15][16][17]. Up to date, there are just a few published procedures where unprotected 3-aminoindoles were isolated and characterized [7,8,18].
On the other hand, common approaches to 3-aminoindole derivatives can be roughly divided into two kinds: non-indolic methods, which build up the aminoindole skeleton from scratch via the Fisher [19,20] or similar multicomponent annulation reactions [7][8][9][10][11][12]14,[21][22][23][24] and post-functionalization procedures based on the corresponding 3-substituted indoles. As for the latter, the major strategies for introducing an amino group at the C3 indole position have remained the nitration [2,13,15,17,25,26] or azidation [18] reactions followed by a reduction to the free amine. The synthesis starts from the corresponding 3-indolecarboxylic acids by a twostep sequence involving the Curtius rearrangement [16,27], palladium-catalyzed amination of indole halides [28], as well as a number of some recent direct C-H amination methods [29][30][31][32][33][34], have been also reported. However, these transformations are mostly multistep processes that require subsequent protection-deprotection or functional group interconversion steps and often suffer from limited scope and efficacy. In turn, herein, we would like to present a novel two-step method for the preparation of unprotected 2-aryl-3-aminoindoles 5 directly from the corresponding 2-aryl indoles 1 and nitrostyrene 2 via intermedial spirocyclic isoxazoles 3 or indolinone 4 (Scheme 1). This approach provides a straightforward synthetic route to otherwise not easily accessible free 3-aminoindoles.

Results
A few years ago, we have discovered [35,36] a somewhat unusual reaction between indoles 6 and nitrostyrenes 7, where the latter act as 1,4-dipoles in the presence of phosphorous acid to give diastereomerically pure spiro-2-indolinone isoxazoles 8 in good to excellent yields (Scheme 2). Subsequent treatment with a mild acid or base leads to 2-(3oxoindolin-2-yl)-2-arylacetonitriles 9 [37,38], which are due to the presence of versatile cyano and carbonyl functional groups could serve as a good synthetic platform for carrying out many other useful transformations. Thus, so far, we have shown that upon the action of KOH in refluxing ethanol, the N-alkyl indolinones 9 formed pyrroloindoles 10, while NH derivatives under the same conditions demonstrated the unexpected extrusion of arylacetonitrile molecule, which eventually result in the formation of hydroxyindolinones 11 [39]. The reduction of 8 or 9 with sodium borohydride has proved to be an efficient way of preparation of corresponding indolylacetamides 12 [40], and a reaction of 2-(3-oxoindolin-2-yl)-2-arylacetonitriles 9 with benzene-1,2-diamines 13 furnished quinoxalines 14 in high-yields [41].
It should be noticed that the latter transformation is accompanied by loss of phenylacetonitrile molecule, so, at some point, we speculated about using hydrazine hydrate instead of o-phenylenediamine derivatives to avoid it. In this case, starting, for instance, from indolinone 4aa, one would expect aminopyridazine 15 as a product analogously to the results of the work [42] (Scheme 3). The authors of that work used the structurally Scheme 1. Two-step C3-amination of 2-arylindoles with nitrostyrene and hydrazine hydrate.

Results
A few years ago, we have discovered [35,36] a somewhat unusual reaction between indoles 6 and nitrostyrenes 7, where the latter act as 1,4-dipoles in the presence of phosphorous acid to give diastereomerically pure spiro-2-indolinone isoxazoles 8 in good to excellent yields (Scheme 2). Subsequent treatment with a mild acid or base leads to 2-(3oxoindolin-2-yl)-2-arylacetonitriles 9 [37,38], which are due to the presence of versatile cyano and carbonyl functional groups could serve as a good synthetic platform for carrying out many other useful transformations.

Results
A few years ago, we have discovered [35,36] a somewhat unusual reaction between indoles 6 and nitrostyrenes 7, where the latter act as 1,4-dipoles in the presence of phosphorous acid to give diastereomerically pure spiro-2-indolinone isoxazoles 8 in good to excellent yields (Scheme 2). Subsequent treatment with a mild acid or base leads to 2-(3oxoindolin-2-yl)-2-arylacetonitriles 9 [37,38], which are due to the presence of versatile cyano and carbonyl functional groups could serve as a good synthetic platform for carrying out many other useful transformations. Thus, so far, we have shown that upon the action of KOH in refluxing ethanol, the N-alkyl indolinones 9 formed pyrroloindoles 10, while NH derivatives under the same conditions demonstrated the unexpected extrusion of arylacetonitrile molecule, which eventually result in the formation of hydroxyindolinones 11 [39]. The reduction of 8 or 9 with sodium borohydride has proved to be an efficient way of preparation of corresponding indolylacetamides 12 [40], and a reaction of 2-(3-oxoindolin-2-yl)-2-arylacetonitriles 9 with benzene-1,2-diamines 13 furnished quinoxalines 14 in high-yields [41].
It should be noticed that the latter transformation is accompanied by loss of phenylacetonitrile molecule, so, at some point, we speculated about using hydrazine hydrate instead of o-phenylenediamine derivatives to avoid it. In this case, starting, for instance, from indolinone 4aa, one would expect aminopyridazine 15 as a product analogously to the results of the work [42] (Scheme 3). The authors of that work used the structurally Thus, so far, we have shown that upon the action of KOH in refluxing ethanol, the N-alkyl indolinones 9 formed pyrroloindoles 10, while NH derivatives under the same conditions demonstrated the unexpected extrusion of arylacetonitrile molecule, which eventually result in the formation of hydroxyindolinones 11 [39]. The reduction of 8 or 9 with sodium borohydride has proved to be an efficient way of preparation of corresponding indolylacetamides 12 [40], and a reaction of 2-(3-oxoindolin-2-yl)-2-arylacetonitriles 9 with benzene-1,2-diamines 13 furnished quinoxalines 14 in high-yields [41].
It should be noticed that the latter transformation is accompanied by loss of phenylacetonitrile molecule, so, at some point, we speculated about using hydrazine hydrate instead of o-phenylenediamine derivatives to avoid it. In this case, starting, for instance, from indolinone 4aa, one would expect aminopyridazine 15 as a product analogously to the results of the work [42] (Scheme 3). The authors of that work used the structurally similar substrate 16 to obtain pyridazinoindole 17 by cyclocondensation, the former with hydrazine hydrate in boiling acetic acid. Keeping that in mind, firstly, we tried to reflux 4aa in hydrazine hydrate, only to get the starting material back unchanged. In the attempt to force the reaction, microwaveassisted heating at 200 • C for 1 h was applied, and this time, the conversion of 4aa did occur, affording, to our surprise, 3-amino-2-phenylindole 5aa as the only isolable product (Scheme 4). In turn, the N-methylated indolinone 18 under the same conditions furnished the corresponding N-Me 3-aminoindole 19, although in a rather modest 41% yield.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 12 similar substrate 16 to obtain pyridazinoindole 17 by cyclocondensation, the former with hydrazine hydrate in boiling acetic acid. Keeping that in mind, firstly, we tried to reflux 4aa in hydrazine hydrate, only to get the starting material back unchanged. In the attempt to force the reaction, microwaveassisted heating at 200 °C for 1 h was applied, and this time, the conversion of 4aa did occur, affording, to our surprise, 3-amino-2-phenylindole 5aa as the only isolable product (Scheme 4). In turn, the N-methylated indolinone 18 under the same conditions furnished the corresponding N-Me 3-aminoindole 19, although in a rather modest 41% yield. Apparently, as opposed to our initial hypothesis, the use of hydrazine hydrate (entry 9) with 4aa does not lead to the target aminopyridazine 15 nor other hydrazines (entries 3-8), and nitrogen binucleophiles (entries 1,2,4-9) lead each time to the 3-aminoindole 5aa (Table 1). Apparently, as opposed to our initial hypothesis, the use of hydrazine hydrate (Entry 9) with 4aa does not lead to the target aminopyridazine 15 nor other hydrazines (Entries 3-8), and nitrogen binucleophiles (Entries 1,2,4-9) lead each time to the 3-aminoindole 5aa (Table 1).
To come up with a plausible mechanism, one had to accommodate all those observations above, namely the formation of the 3-aminoindole 5aa with both R 3 CH 2 NH 2 (Entries 1,2) and hydrazine (Entries 3-9) derivatives while lacking any expected products in the case of hydroxylamine (Entry 10). We speculate (Scheme 5) that for the amine-bearing α-CH 2 group (such as ethylene or 1,2-propylenediamines), the condensation of the latter with the starting indolinone A leads to the corresponding imine B. The following proton abstraction from the N-alkylimino fragment, accompanied by simultaneous benzyl cyanide loss, gives a new, aromatization-driven imine C. Then, due to an excess of R 3 CH 2 NH 2 , a recondensation occurs, resulting in the target aminated indole D. In favor of this mechanism, a key feature of which is the presence of α-CH protons, speaks to our previous finding [41], where a reaction of (3-oxoindolin-2-yl)acetonitriles A with 1,2-phenylenediamines ends up in quinoxalines 14 (Scheme 2). If anything like that were to happen in the case of 1,2-diamines (Entries 1,2), then the formation of the corresponding dihydroquinaxolines was expected but did not occur. Apparently, as opposed to our initial hypothesis, the use of hydrazine hydrate (entry 9) with 4aa does not lead to the target aminopyridazine 15 nor other hydrazines (entries 3-8), and nitrogen binucleophiles (entries 1,2,4-9) lead each time to the 3-aminoindole 5aa (Table 1). To come up with a plausible mechanism, one had to accommodate all those observations above, namely the formation of the 3-aminoindole 5aa with both R3CH2NH2 (Entries 1,2) and hydrazine (Entries 3-9) derivatives while lacking any expected products in the case of hydroxylamine (Entry 10). We speculate (Scheme 5) that for the amine-bearing α-CH2 group (such as ethylene or 1,2-propylenediamines), the condensation of the latter with the starting indolinone A leads to the corresponding imine B. The following proton abstraction from the N-alkylimino fragment, accompanied by simultaneous benzyl cyanide loss, gives a new, aromatization-driven imine C. Then, due to an excess of R3CH2NH2, a recondensation occurs, resulting in the target aminated indole D. In favor of this mechanism, a key feature of which is the presence of α-CH protons, speaks to our previous finding [41], where a reaction of (3-oxoindolin-2-yl)acetonitriles A with 1,2-phenylenediamines ends up in quinoxalines 14 (Scheme 2). If anything like that were to happen in the case of 1,2-diamines (Entries 1,2), then the formation of the corresponding dihydroquinaxolines was expected but did not occur. Scheme 5. Proposed mechanism of 3-aminoindoles formation with R3CH2NH2-type nucleophiles.
Meanwhile, in the case of hydrazine derivatives (entries 3-9), supposedly, the reaction takes a slightly different pathway (Scheme 6). First of all, like with imine B (Scheme 5), the formation of hydrazone E should occur. The following extrusion of a phenylacetonitrile molecule would lead to the azo-heteroarene F. As shown previously, the -N=N-bond undergoes a reductive cleavage to the corresponding anilines upon the action of (among other reductants) hydrazine hydrate in the presence of, for instance, Raney Ni [43], aluminum powder [44], or even without any catalyst by simple heating in ethanol [45]. We assume that something similar takes place in our case resulting eventually in the target 3-aminoindole D. Scheme 6. Proposed mechanism of 3-aminoindoles formation with R3NHNH2-type nucleophiles.
Lastly, in the reaction with hydroxylamine (entry 10), no meaningful products were isolated. However, the N-methyl derivative 18, under the same conditions, unexpectedly gave 1-methyl-2-phenylindole J, although with a low 15% yield (Scheme 7). Most likely, the corresponding oxime G, after the loss of the BnCN molecule, became nitrosoindole H. Further hydrolysis of benzyl cyanide and its condensation with the nitroso group of indole H produced the azo compound I, which in turn got reduced in some Wolff-Kishner-type reaction to form the 1-methyl-2-phenylindole J. Scheme 5. Proposed mechanism of 3-aminoindoles formation with R 3 CH 2 NH 2 -type nucleophiles.
Meanwhile, in the case of hydrazine derivatives (Entries 3-9), supposedly, the reaction takes a slightly different pathway (Scheme 6). First of all, like with imine B (Scheme 5), the formation of hydrazone E should occur. The following extrusion of a phenylacetonitrile molecule would lead to the azo-heteroarene F. As shown previously, the -N=N-bond undergoes a reductive cleavage to the corresponding anilines upon the action of (among other reductants) hydrazine hydrate in the presence of, for instance, Raney Ni [43], aluminum powder [44], or even without any catalyst by simple heating in ethanol [45]. We assume that something similar takes place in our case resulting eventually in the target 3-aminoindole D. To come up with a plausible mechanism, one had to accommodate all those observations above, namely the formation of the 3-aminoindole 5aa with both R3CH2NH2 (Entries 1,2) and hydrazine (Entries 3-9) derivatives while lacking any expected products in the case of hydroxylamine (Entry 10). We speculate (Scheme 5) that for the amine-bearing α-CH2 group (such as ethylene or 1,2-propylenediamines), the condensation of the latter with the starting indolinone A leads to the corresponding imine B. The following proton abstraction from the N-alkylimino fragment, accompanied by simultaneous benzyl cyanide loss, gives a new, aromatization-driven imine C. Then, due to an excess of R3CH2NH2, a recondensation occurs, resulting in the target aminated indole D. In favor of this mechanism, a key feature of which is the presence of α-CH protons, speaks to our previous finding [41], where a reaction of (3-oxoindolin-2-yl)acetonitriles A with 1,2-phenylenediamines ends up in quinoxalines 14 (Scheme 2). If anything like that were to happen in the case of 1,2-diamines (Entries 1,2), then the formation of the corresponding dihydroquinaxolines was expected but did not occur.

Scheme 5. Proposed mechanism of 3-aminoindoles formation with R3CH2NH2-type nucleophiles.
Meanwhile, in the case of hydrazine derivatives (entries 3-9), supposedly, the reaction takes a slightly different pathway (Scheme 6). First of all, like with imine B (Scheme 5), the formation of hydrazone E should occur. The following extrusion of a phenylacetonitrile molecule would lead to the azo-heteroarene F. As shown previously, the -N=N-bond undergoes a reductive cleavage to the corresponding anilines upon the action of (among other reductants) hydrazine hydrate in the presence of, for instance, Raney Ni [43], aluminum powder [44], or even without any catalyst by simple heating in ethanol [45]. We assume that something similar takes place in our case resulting eventually in the target 3-aminoindole D. Scheme 6. Proposed mechanism of 3-aminoindoles formation with R3NHNH2-type nucleophiles.
Lastly, in the reaction with hydroxylamine (entry 10), no meaningful products were isolated. However, the N-methyl derivative 18, under the same conditions, unexpectedly gave 1-methyl-2-phenylindole J, although with a low 15% yield (Scheme 7). Most likely, the corresponding oxime G, after the loss of the BnCN molecule, became nitrosoindole H. Further hydrolysis of benzyl cyanide and its condensation with the nitroso group of indole H produced the azo compound I, which in turn got reduced in some Wolff-Kishner-type reaction to form the 1-methyl-2-phenylindole J. Scheme 6. Proposed mechanism of 3-aminoindoles formation with R 3 NHNH 2 -type nucleophiles.
Lastly, in the reaction with hydroxylamine (Entry 10), no meaningful products were isolated. However, the N-methyl derivative 18, under the same conditions, unexpectedly gave 1-methyl-2-phenylindole J, although with a low 15% yield (Scheme 7). Most likely, the corresponding oxime G, after the loss of the BnCN molecule, became nitrosoindole H. Further hydrolysis of benzyl cyanide and its condensation with the nitroso group of indole H produced the azo compound I, which in turn got reduced in some Wolff-Kishner-type reaction to form the 1-methyl-2-phenylindole J. Next, we evaluated the scope and limitations of the described procedure (entry 9). For that, a series of indolinones 4 bearing various aryl substituents R1 was introduced into the reaction with hydrazine hydrate under the chosen conditions (Method A). As seen in Scheme 7. Plausible mechanism of formation of 1-methyl-2-phenylindole J.
Next, we evaluated the scope and limitations of the described procedure (Entry 9). For that, a series of indolinones 4 bearing various aryl substituents R 1 was introduced into the reaction with hydrazine hydrate under the chosen conditions (Method A). As seen in Scheme 8, all these substrates reacted smoothly, producing the corresponding products 5aa-5ag in good to high yields. The presence of alkyl or fluorine substitutes at C-5 in the indoline core did not affect the reaction performance, and the target 5-substituted 3aminoindoles 5ab, 5ac, and 5ad were also obtained in high yields (Scheme 8). Remarkably, the direct conversion of spiranes 3 into aminoindoles 5 (Method B) is also possible, giving yields comparable to those obtained via Method A. Scheme 7. Plausible mechanism of formation of 1-methyl-2-phenylindole J.
Next, we evaluated the scope and limitations of the described procedure (entry 9). For that, a series of indolinones 4 bearing various aryl substituents R1 was introduced into the reaction with hydrazine hydrate under the chosen conditions (Method A). As seen in Scheme 8, all these substrates reacted smoothly, producing the corresponding products 5aa-5ag in good to high yields. The presence of alkyl or fluorine substitutes at C-5 in the indoline core did not affect the reaction performance, and the target 5-substituted 3-aminoindoles 5ab, 5ac, and 5ad were also obtained in high yields (Scheme 8). Remarkably, the direct conversion of spiranes 3 into aminoindoles 5 (Method B) is also possible, giving yields comparable to those obtained via Method A. Scheme 8. Unprotected 2-aryl-3-aminoindoles prepared by described herein procedure.
Finally, we tested the possibility of subsequent protection of the newly formed amino group by running a reaction in the mixture of hydrazine hydrate and acetic acid. Expectedly, the corresponding N-acyl derivatives N-Ac 5aa, 5ai were received, although in somewhat reduced yields (Scheme 9). Scheme 8. Unprotected 2-aryl-3-aminoindoles prepared by described herein procedure.
Finally, we tested the possibility of subsequent protection of the newly formed amino group by running a reaction in the mixture of hydrazine hydrate and acetic acid. Expectedly, the corresponding N-acyl derivatives N-Ac 5aa, 5ai were received, although in somewhat reduced yields (Scheme 9).
In the end, we would like to share some of our thoughts and observations regarding the shelf stability of the described herein free 3-aminoindoles. It seems that there is a general consensus about the sensitivity of these substances to air and light both in solution and solid and, as a result, the inability to purify them by column chromatography [7,8]. At the same time, we were able to work with most of them (but not sample 19) rather comfortably, including short and fast (10-15 min) column purifications on silica. The freshly prepared samples, usually light grey or beige, indeed became deep blue with time but even then, their proton NMR spectra showed no signs of significant decomposition. Additionally, while we did not measure the life length of our samples specifically, it might be that the commonly thought tendency of 3-aminoindoles toward oxidative breakdown is somewhat overrated. In the end, we would like to share some of our thoughts and observations regarding the shelf stability of the described herein free 3-aminoindoles. It seems that there is a general consensus about the sensitivity of these substances to air and light both in solution and solid and, as a result, the inability to purify them by column chromatography [7,8]. At the same time, we were able to work with most of them (but not sample 19) rather comfortably, including short and fast (10-15 min) column purifications on silica. The freshly prepared samples, usually light grey or beige, indeed became deep blue with time but even then, their proton NMR spectra showed no signs of significant decomposition. Additionally, while we did not measure the life length of our samples specifically, it might be that the commonly thought tendency of 3-aminoindoles toward oxidative breakdown is somewhat overrated.

General Information
NMR spectra, 1 H, 13 C, and 19 F were measured in solutions of CDCl3 or DMSO-d6 on Bruker AVANCE-III HD instrument (at 400, 101, and 376 MHz, respectively). Residual solvent signals were used as internal standards in DMSO-d6 (2.50 ppm for 1 H and 40.45 ppm for 13 С nuclei) or CDCl3 (7.26 ppm for 1 H and 77.16 ppm for 13 С nuclei). HRMS spectra were measured on Bruker maXis impact (electrospray ionization in MeCN solutions, employing HCO2Na-HCO2H for calibration). IR spectra were measured on FT-IR spectrometer Shimadzu IRAffinity-1S equipped with an ATR sampling module. Spectral data are provided in the Supplementary Materials (S1-S42). Reaction progress, purity of isolated compounds, and Rf values were monitored with TLC on Silufol UV-254 plates. Column chromatography was performed on silica gel (32-63 μm, 60 Å pore size). Melting points were measured with Stuart SMP30 apparatus. All spirocyclic indoles 3 and indolinones 4, except 4ab and 4ad, were synthesized according to the previously reported procedures and were identical to those described [37]. All reagents and solvents were purchased from commercial vendors and used as received.

General Information
NMR spectra, 1 H, 13 C, and 19 F were measured in solutions of CDCl 3 or DMSO-d 6 on Bruker AVANCE-III HD instrument (at 400, 101, and 376 MHz, respectively). Residual solvent signals were used as internal standards in DMSO-d 6 (2.50 ppm for 1 H and 40.45 ppm for 13 C nuclei) or CDCl 3 (7.26 ppm for 1 H and 77.16 ppm for 13 C nuclei). HRMS spectra were measured on Bruker maXis impact (electrospray ionization in MeCN solutions, employing HCO 2 Na-HCO 2 H for calibration). IR spectra were measured on FT-IR spectrometer Shimadzu IRAffinity-1S equipped with an ATR sampling module. Spectral data are provided in the Supplementary Materials (S1-S42). Reaction progress, purity of isolated compounds, and R f values were monitored with TLC on Silufol UV-254 plates. Column chromatography was performed on silica gel (32-63 µm, 60 Å pore size). Melting points were measured with Stuart SMP30 apparatus. All spirocyclic indoles 3 and indolinones 4, except 4ab and 4ad, were synthesized according to the previously reported procedures and were identical to those described [37]. All reagents and solvents were purchased from commercial vendors and used as received. These compounds were prepared in analogy to the method described in [37]. A Weaton microreactor equipped with magnetic spin-vane and Mininert valve was charged with a mixture of (E)-(2-Nitrovinyl)benzene (150 mg, 1.0 mmol), corresponding 5-halide-2-phenyl-1H-indole (1.0 mmol), phosphorus acid (1.0 g), and formic acid (1.0 g). The mixture was vigorously stirred for 2 h at room temperature while it turned dark red and homogenized. Then, the mixture was poured into water (50 mL), and the formed crude spirane 3 precipitate was collected and washed with water (4 × 20 mL), dried, and dissolved in ethanol (4 mL). Triethylamine (102 mg, 1.0 mmol) was added, and the resulting solution was stirred at room temperature for 3 h. Crystalline precipitate of crude product was formed, which was collected and purified by preparative column chromatography on silica gel, eluting with ethyl acetate/hexane mixture (1:4).

Conclusions
A novel preparative method for the synthesis of diverse 3-aminoindoles 5 based on a microwave-assisted cascade reaction of 2-(3-oxoindolin-2-yl)-2-arylacetonitriles 4 with hydrazine hydrate was developed. Alternatively, the same transformation could also be carried out from 4 -phenyl-4 H-spiro[indole-3,5 -isoxazoles] 3. Considering that such spirocyclic indoles 3 could be obtained in a single step from commonly available indoles 1 and nitrostyrene 2, the overall sequence provides a very convenient and affordable route to generally not easily available 3-aminoindoles.